This proposal deals with the inhibition of cysteine proteases by boronic acid transition state analogs. Boronic acids are well-known inhibitors of serine and metallo-hydrolases. They have not been studied in relation to thiol enzymes. Earlier attempts to observe such inhibitions had failed. In preliminary work done recently in this laboratory, it has been shown that the thiol protease cathepsin C (dipeptidyl peptidase I, E.C. 3.4.14.1) is inhibited by arylboronic acids. We intend to study the inhibition of thiol proteases by boronic acid analogs of arginine and phenylalanine. The pH-dependence of inhibition for these enzymes combined with similar data already available for the serine proteases is expected to help show how thiol and serine proteases differ in their mechanisms of acylation and deacylation. The enzymes used will be papain an extensively studied thiol protease with an argining specificity at the S1 subsite, and cathepsin C, a less-well studied thiol protease with a specificity for aromatic amino acids in the S1 subsite. This study will involve the chemical synthesis of peptide boronic acids: Gly-BoroPhe for cathepsin C, and Ala-Ala-Phe-BoroArg for papain. This will be followed by a steady-state kinetic investigation of the effects of these compounds on the appropriate enzyme at different pH values. (BoroPhe is the boronic acid analog of phenylalanine, with boron substituting for the carboxylic acid carbon atom; BoroArg is the analogous arginine derivative.) The inhibition of thiol proteases by small-molecule inhibitors is a biologically interesting topic since it has been shown that peptide aldehydes, which inhibit both serine and thiol proteases, delay the onset of muscle deterioration in animal models of muscular dystrophy. This disease is associated with elevated levels of muscle proteinases, including cathepsin C. Therefore, specific inhibitors of cathepsin C may be agents useful in the study of this disease. This study includes the first preparation of peptides containing BoroArg. Successful synthesis of such peptides will make it possible, in the longer term, to study their interaction with biologically interesting trypsin-like serine proteases. Examples are the enzymes of the blood-clotting cascade, as well as plasmin and plasminogen activator.